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Journal of Bacteriology, August 2000, p. 4625-4627, Vol. 182, No. 16
Department of Biochemistry, School of
Pharmacy, University of Barcelona, 08028 Barcelona, Spain
Received 12 February 2000/Accepted 2 June 2000
Genes yiaP and yiaR of the
yiaKLMNOPQRS cluster of Escherichia coli are
required for the metabolism of the endogenously formed L-xylulose, whereas yiaS is required for this
metabolism only in araD mutants. Like AraD, YiaS was shown
to have L-ribulose-5-phosphate 4-epimerase activity.
Similarity of YiaR to several 3-epimerases suggested that this protein
could catalyze the conversion of L-xylulose-5-phosphate into L-ribulose-5-phosphate, thus completing the pathway
between L-xylulose and the general metabolism.
On the basis of the similarity
displayed by some of the gene products of the yiaKLMNOPQRS
(yiaK-S) gene cluster (accession no. U00039) Sofia et al.
(14) have proposed that this cluster could be involved in
the metabolism of carbohydrates, although the precise substrate has not
been determined. More recently, analyses in silico of the corresponding
protein sequences by Reizer et al. (11) have permitted the
comparison with other known proteins and the suggestion of putative
biochemical functions for some of them.
In another context, the characterization of the metabolic pathway
allowing the utilization of L-lyxose by mutants of
Escherichia coli has shown the presence of a gene encoding a
new highly specific L-xylulose kinase (12).
L-Xylulose was formed in these mutants by the action of
L-rhamnose isomerase on L-lyxose. The
restriction map of the region encompassing this gene has permitted its
identification as the gene yiaP of the yiaK-S
operon. To specify if some other genes among the nine belonging to the
yiaK-S operon (Fig. 1) were involved in L-lyxose metabolism, complementation analysis
of L-lyxose-negative strains with plasmids containing
different fragments of this gene cluster was performed.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of the yiaR and yiaS
Genes of Escherichia coli in Metabolism of Endogenously
Formed L-Xylulose
ABSTRACT
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FIG. 1.
Genetic map of the region encompassing the
yiaK-S operon and complementation analysis of different
yiaK-S mutants. The open bar represents the genomic DNA of
strain JA134 and the arrows represent the extension and direction of
the transcribed yiaK-S genes. Insertions of the CAT cassette
are indicated by black bars below the map and are labeled by the name
of the mutant strain carrying them. Thick lines correspond to the
different inserts of the plasmids used in the complementation
experiments whose results are shown in the contiguous table. Thin lines
show the deleted fragment in the corresponding plasmid. A,
AgeI; B, BamHI; Bg, BglII; Bs,
BstXI; H, HindIII; P, PstI; S,
SalI; V, EcoRV; X, XhoI; Xc,
XcmI.
L-Lyxose phenotypic complementation of yiaQ, yiaR, and yiaS mutants. Several mutants unable to utilize L-lyxose were derived from strain JA134 (12) by ethyl methanesulfonate mutagenesis (10), growth on rich medium (3), and selection by replica plating on minimal medium (3) with L-lyxose or glucose. The presence of L-xylulose kinase activity, measured as described previously (12), in some of the mutants indicated that their negative phenotype was due to impairment of a gene function other than L-xylulose kinase. Mutations in the L-rhamnose permease or isomerase required for L-lyxose metabolism (2, 12) were ruled out by analyzing the ketose excretion (7) when grown on L-rhamnose. After this screening, strain JA192 was selected for further analysis.
Previous experiments had shown that L-lyxose utilization did not require genes of the yiaK-S system other than those included in plasmid pJC1 (12). Partial fragments of the insert present in pJC1 were cloned in pBR322 vector, and the derivative plasmids (Fig. 1) were used in complementation analysis of strain JA192. The mutation yielding the L-lyxose-negative phenotype was located in the yiaR gene, since it was complemented by every plasmid except pJC2, pJTP5, or pJTP7 (Fig. 1). To study whether the yiaQ and yiaS gene products are involved in L-lyxose metabolism and to confirm the yiaR participation, chromosomal null mutations in each of these genes were obtained by insertional mutation of the CAT19 cassette (8) and subsequent transference, first to strain JC7623 (15) and then to our genetic background according to the method of Winans et al. (16). When the knockout constructs of yiaP, yiaQ, and yiaR were transferred to the JA134 background (strains JA193, 194, and JA195, respectively), the cells became L-lyxose negative (Fig. 1). The knockout construct of yiaS (strain JA196) did not affect the L-lyxose phenotype. Complementation of strain JA195 (yiaR::cat) by plasmids carrying the fragment in which the insertion was located confirmed the participation of yiaR in L-lyxose metabolism. Complementation of strain JA194 (yiaQ::cat) by plasmids carrying only yiaR indicated that chloramphenicol acetyltransferase (CAT) insertion in yiaQ exerted polarity effects on yiaR and that the yiaQ gene was not necessary for L-lyxose utilization. Furthermore, growth of wild-type strain ECL1 (9) on L-lyxose when transformed with plasmid pTP4 lacking yiaQ again showed that this gene product was not required for L-lyxose metabolism.yiaR and yiaS gene product analysis. Computational analysis of the gene product encoded by yiaS yielded 76% identity over 231 amino acids with the L-ribulose-5-phosphate 4-epimerase of Escherichia coli (product of araD gene), which catalyzes an early step in L-arabinose utilization and 74% identity over 203 amino acids with that of Salmonella enterica serovar Typhimurium. Thus, the yiaS gene product is assigned to the AraD-FucA family.
Plasmids carrying yiaS (Fig. 1) allowed the strain MC4100 (5), reported to be an araD mutant, to utilize L-arabinose, indicating that the yiaS gene complemented araD function and that its product utilized L-ribulose-5-phosphate as a substrate. In addition, Northern blot experiments indicated that L-lyxose was able to induce the ara operon transcription in strain JA134 (not shown). For these experiments, total RNA was obtained from cultures of strain JA134 in casein acid hydrolysate in the absence or in the presence of L-lyxose. An araD gene fragment obtained by PCR with primers P1 (GGCGCTAACTGACGGCAG) and P2 (TAGAAGATCTCAAACGCC) was used as a probe. The induction of the ara system by L-lyxose and the high similarity displayed by YiaS and AraD suggest that AraD could complement the YiaS function in strain JA196. To determine whether AraD complemented YiaS function, the araD mutation of strain MC4100 was transferred into the genetic background of strain JA196 (yiaS::cat) by the following strategy. (i) A Tn10 marker was transduced from strain CAG12095 (13) into strain MC4100, and tetracycline-resistant transductants that retained the inability to utilize L-arabinose were selected. (ii) One of these transductants (strain JA197) was subsequently used to transduce the two linked markers into strain JA196. A double mutant (strain JA198) was isolated and transformed with plasmids containing yiaS or araD and tested for its ability to utilize both L-lyxose and L-arabinose. None of the transformants analyzed displayed impairment in either of these metabolisms, showing that the 4-epimerase function could be supplied by either of the two YiaS and AraD proteins. To test if L-lyxose itself or an intermediate metabolite was the inducer of the arabinose system, a Tn5 insertion mutant in the rhaA gene (encoding rhamnose isomerase) was obtained from strain JA134 (strain JA199) by using phage
467 (b221 cIts857 rex::Tn5
Oam29 Pam80) (4). This mutant was selected
by its inability to utilize L-lyxose and the inability to
excrete L-rhamnulose when grown in the presence of
L-rhamnose. The precise location of the insertion in
rhaA was assessed by cloning and sequencing the chromosomal
region close to the Tn5 by using an internal sequence of the
transposon as a primer. Induction of the ara operon by L-lyxose in strain JA199 indicated that this compound was
directly activating the arabinose operon expression.
To confirm the L-ribulose-5-phosphate 4-epimerase
catalytic activity encoded by the yiaS gene, the
experimental procedure described by Deupree and Wood (6) was
set up. To this end, araB (encoding L-ribulose
kinase), araD, and yiaS genes of E. coli were cloned into plasmid pUC19. Overexpression was determined for araB and araD by enzyme activity assays in
extracts of strain ECL1 transformed with the corresponding plasmid
grown on casein acid hydrolysate. The
D-xylulose-5-phosphate formed when L-ribulose was added to a reaction mixture containing extracts of cells
overexpressing araB and yiaS yielded an oxidation
of NADH of 190 to 210 nmol/min/mg. No significant consumption of NADH
was detected when araB or yiaS gene products were
lacking. As a positive control, we have run in parallel a reaction with
overexpressed araD instead of yiaS.
To test whether the yiaS gene product could recognize
L-xylulose-5-phosphate as a substrate, another experiment
was conducted coupling this gene product with commercial
D-ribulose-5-phosphate 3-epimerase, which should yield
D-xylulose-5-phosphate (Fig.
2). All attempts were unsuccessful,
proving that the yiaS gene product did not convert
L-xylulose-5-phosphate into
D-ribulose-5-phosphate.
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The yiaK-S operon is involved in the metabolism of endogenous L-xylulose. Although the constitutive expression of the yiaK-S gene cluster allowed strain JA134 to metabolize L-xylulose generated endogenously from L-lyxose by the action of L-rhamnose isomerase, this strain was unable to efficiently utilize this ketose as a sole source of carbon and energy. Furthermore, the same behavior was observed with wild-type strain ECL1 transformed with plasmid pJC1 carrying the genes required for L-xylulose metabolism. These results suggested the inability of these cells to uptake external L-xylulose. Given the structural similarity between L-lyxose, known to be transported by L-rhamnose permease (2), and L-xylulose, competition of this ketose with L-rhamnose for L-rhamnose permease was studied. The results indicate that L-rhamnose permease does not recognize L-xylulose, in contrast to the effect observed with L-lyxose, which produced a strong inhibition of the L-rhamnose transport (data not shown). Inefficient L-xylulose transport suggests that L-xylulose has to be generated endogenously from L-lyxose or in the processing of a hitherto unknown carbohydrate.
It is likely that the yiaK-S cluster evolved for the metabolism of carbohydrate(s) other than L-lyxose, since not all the genes in the operon are involved in the metabolism of this pentose. In order to establish the function of the yiaK-S operon, it is instructive to consider that of the nine gene products of the yia operon, only three, the kinase previously reported (12) and YiaR and YiaS studied in this work, catalyzed the steps required to convert L-xylulose into a D-xylulose-5-phosphate which enters the pentose phosphate pathway. The remaining yia operon genes must be responsible for the generation of this intermediate from an internal or external precursor. Although we demonstrated that L-lyxose is an inducer of the ara operon, it is unlikely that this operon could be induced by the unknown physiological substrate. Therefore, YiaS would not be complemented by AraD and would result in an essential function for its metabolism.| |
ACKNOWLEDGMENTS |
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This work was supported by grant PB97-0920 from the Dirección General de Enseñanza Superior e Investigación Científica, Madrid, Spain, and partially by the help of the "Comissionat per Universitats i Recerca de la Generalitat de Catalunya." E.I. is the recipient of a predoctoral fellowship from the Generalitat de Catalunya.
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FOOTNOTES |
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* Corresponding author. Mailing address: Departamento de Bioquímica, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain. Phone: 34-93-402 4521. Fax: 34-93-402 1896. E-mail: Palaciu{at}farmacia.far.ub.es.
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Journal of Bacteriology, August 2000, p. 4625-4627, Vol. 182, No. 16
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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